ABSTRACT Recently, genome-wide association studies have implicated the human LIN28B locus in regulating height and the timing of menarche. LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators of mammalian body size and metabolism. To explore the function of the Lin28-Let-7 pathway in vivo, we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. Investigation of metabolic and endocrine mechanisms of overgrowth in these transgenic mice revealed increased glucose metabolism and insulin sensitivity. Here we report a mouse that models the human phenotypes associated with genetic variation in the Lin28-Let-7 pathway.

[Show abstract][Hide abstract]ABSTRACT: Mammals exhibit a remarkable variety of phenotypes and comparative studies using novel model species are needed to uncover the evolutionary developmental mechanisms generating this diversity. Here, we undertake a developmental biology and numerical modeling approach to investigate the development of skin appendages in the spiny mouse, Acomys dimidiatus.
We demonstrate that Acomys spines, possibly involved in display and protection, are enlarged awl hairs with a concave morphology. The Acomys spines originate from enlarged placodes that are characterized by a rapid downwards growth which results in voluminous follicles. The dermal condensation (dermal papilla) at the core of the follicle is very large and exhibits a curved geometry. Given its off-centered position, the dermal papilla generates two waves of anisotropic proliferation, first of the posterior matrix, then of the anterior inner root sheath (IRS). Higher in the follicle, the posterior and anterior cortex cross-section areas substantially decrease due to cortex cell elongation and accumulation of keratin intermediate filaments. Milder keratinization in the medulla gives rise to a foamy material that eventually collapses under the combined compression of the anterior IRS and elongation of the cortex cells. Simulations, using linear elasticity theory and the finite-element method, indicate that these processes are sufficient to replicate the time evolution of the Acomys spine layers and the final shape of the emerging spine shaft.
Our analyses reveal how hair follicle morphogenesis has been altered during the evolution of the Acomys lineage, resulting in a shift from ancestral awl follicles to enlarged asymmetrical spines. This study contributes to a better understanding of the evolutionary developmental mechanisms that generated the great diversity of skin appendage phenotypes observed in mammals.

[Show abstract][Hide abstract]ABSTRACT: Human height is a highly variable trait, both within and between populations, has a high heritability, and influences the manner in which people behave and are treated in society. Although we know much about human height, this information has rarely been brought together in a comprehensive, systematic fashion. Here, we present a synthetic review of the literature on human height from an explicit evolutionary perspective, addressing its phylogenetic history, development, and environmental and genetic influences on growth and stature. In addition to presenting evidence to suggest the past action of natural selection on human height, we also assess the evidence that natural and sexual selection continues to act on height in contemporary populations. Although there is clear evidence to suggest that selection acts on height, mainly through life-history processes but perhaps also directly, it is also apparent that methodological factors reduce the confidence with which such inferences can be drawn, and there remain surprising gaps in our knowledge. The inability to draw firm conclusions about the adaptiveness of such a highly visible and easily measured trait suggests we should show an appropriate degree of caution when dealing with other human traits in evolutionary perspective.

[Show abstract][Hide abstract]ABSTRACT: Lin28B, a homologue of Lin28, represses biogenesis of let-7 microRNAs with a role in tumorigenesis and is considered a potential therapeutic target for various human cancers. The aim of the study was to identify the clinical significance of Lin28B in gastric adenocarcinoma (GAC). We examined the expression of Lin28B in 97 human gastric cancer samples with 32 samples of non-dysplastic tissues by immunohistochemistry. In the 97 GAC cases, 42 were with high Lin28B expression. The expression levels of Lin28B proteins in GAC were higher than in corresponding adjacent normal tissues (P=0.001). Significant correlations were noted between Lin28B expression and lymph node status (P=0.005), TNM stage (P < 0.001), tumor invasion (P=0.036), and differentiation (P=0.001) of GAC patients. The Kaplan-Meier estimates showed a negative correlation of overall 5-year survival rate with Lin28B expression where higher expression resulted in poorer prognosis in GAC. In univariate analysis, lymph node metastasis, TNM stage, serosal invasion, Lin28B expression as well as differentiation grade could predict the prognosis of GAC patients (P=0.002, P < 0.001, P=0.003, P < 0.001, P=0.001, respectively). Multivariate analysis revealed that the expression level of Lin28B (P < 0.001), TNM stage (P < 0.001) as well as differentiation grade (P < 0.001) were the three potential independent prognostic factors in our study [Hazard ratio (HR)=2.108 and P=0.017, HR=1.994 and P=0.018, HR=1.939 and P=0.046, respectively]. Our findings point to the prognostic role of Lin28B in GAC, and indicate Lin28B as a potential therapeutic target of GAC patients.

International journal of clinical and experimental pathology. 01/2014; 7(8):5083-92.

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13Howard Hughes Medical Institute, Boston, Massachusetts, USA.14Manton Center for Orphan Disease Research, Boston, Massachusetts, USA.AbstractRecently, genome-wide association studies (GWAS) have linked the human LIN28B locus toheight and timing of menarche [1-5]. LIN28B and its homolog LIN28 (hereafter, LIN28A) arefunctionally redundant RNA-binding proteins that block let-7 microRNA (miRNA) biogenesis[6-9]. lin-28 and let-7 were discovered in C. elegans as heterochronic regulators of larval andvulval development, but recently have been implicated in cancer, stem cell aging, andpluripotency [10-13]. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators ofmammalian body size and metabolism [14-18]. To explore the Lin28/let-7 pathway in vivo, weengineered transgenic mice to express Lin28a and observed increased body size, crown-rumplength, and a delayed onset of puberty. While investigating metabolic and endocrine mechanismsof overgrowth, we observed increased glucose metabolism and insulin sensitivity in thesetransgenic mice. We report a mouse that models the human phenotypes associated with geneticvariation in the Lin28/let-7 pathway.To investigate Lin28a function in vivo, we generated a transgenic mouse strain thatexpresses the M2 reverse tetracycline transactivator (M2-rtTA) from the Rosa26 locus andFlag-tagged mouse Lin28a from the Collagen 1a1 (Col1a1) locus (Fig. 1a) [19]. Weobserved that mice carrying the Lin28a transgene (Lin28a Tg) were larger than non-transgenic littermates, even in the absence of the rtTA transgene and/or doxycyclineinduction. Transgene-bearing animals also had wider facies and coarser hair (Fig. 1b).Lin28a Tg animals showed an increased growth rate and in adulthood were heavier andlonger (Fig. 1c-d). Newborn wild-type (WT) and Lin28a Tg pups were the same size (Fig.S1a). Differences in weight and crown-rump length became apparent after weaning, and theincreased growth of Tg mice was characterized by a prolonged growth period with higherplateaus for height and weight. In contrast, we found that Lin28a knockout mice weigh 20%less at birth than WT pups, but do not survive long enough for further analysis (Fig. S1b).DEXA imaging revealed no change in percentage body fat or lean mass in Lin28a Tg mice,but did show increased bone mineral content and density (Fig. 1e). Organ mass wasincreased in relative proportion to total body weight, suggesting appropriate regulation oforgan size relative to overall body growth (Fig. 1f). To control for any possible effects ofdoxycycline induction of the engineered Col1a1 locus, we generated a mouse strain usingthe KH2 embryonic stem cell (ESC) line without a targeted transgene. After 5 weeks ofdoxycycline, the WT, induced, and un-induced mice containing the engineered Col1a1 alleleshowed no differences in weight (Fig. S1c). Taken together, our data show that Lin28a Tgmice possess increased body size, a phenotype associated with genetic variation in thehuman LIN28B locus.Given the recent GWAS linking LIN28B to later age at menarche, we investigated thetiming of reproductive maturity in these mice. Vaginal opening (VO) is a key milestone insexual development and a reliable marker for the onset of murine puberty [20]. In Lin28a Tgmice, we observed a 2.24 and 2.18 day delay in VO in the CD-1 and C57BL/6J strains,respectively (Table 1 and Fig. 2a-b; both p < 0.002). Puberty was delayed despite the factthat Lin28a Tg mice were heavier at VO for both strain backgrounds (Table 1 and Fig. 2c;both p < 0.0001). At day 26 of age, uterine plus ovarian mass was greater in the WT mice,indicating delayed sexual development in Tg mice (Fig. 2d; p < 0.002). WT and Lin28a Tgmice achieved first estrus at day 27.3 and day 31.8, respectively (Fig. 2e; p = 0.0106).Furthermore, mating experiments revealed that Lin28a Tg animals had a ~3 day delay toZhu et al.Page 2Nat Genet. Author manuscript; available in PMC 2011 April 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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date of first litter (Fig. 2f; p = 0.0351), correlating well with the delays in VO and firstestrus. We noted that the size of the first litter was markedly larger in the Tg versus WTanimals (16.4 vs. 9.67; p = 0.0002) (Fig. 2g), though overall fertility was no different overthe first three months of life, indicating that altered timing of sexual maturation was not dueto reproductive incompetence.We then analyzed Lin28a transgene and let-7 expression in various organs. Normally, bothLin28a and Lin28b are highly expressed in early murine embryogenesis until E10.5.Thereafter, their patterns diverge: Lin28b is expressed in the fetal liver, blood, and brain,while Lin28a is expressed in the intestinal crypts, muscle, heart, testes, and ovary [21]. ByRT-PCR, combined transgene and endogenous Lin28a mRNA expression was increased inmultiple organs in the Tg mice, resulting from ectopic expression of the Lin28a transgenefrom the Col1A1 locus in both neonates and adults (Fig. 3a-b). In adults, there was a 7-foldLin28a mRNA increase in the skeletal muscle, a 5-fold increase in the ovary, and a 4-foldincrease in the hypothalamus (Fig. 3a). In the neonatal limb, which contains skin andmuscle, there was a 3-fold increase (Fig. 3b). These fold changes were small, consideringthat endogenous Lin28a is expressed in these tissues. In the muscle, skin and neonatal limb,the additional Lin28a protein functioned to suppress let-7 processing (Fig. 3c-d). In contrast,let-7 levels were preserved in the liver, where Lin28a is absent (Fig. 3c-d). To corroboratethese results, immunohistochemistry (IHC) was used to show increased Lin28a protein inskin and muscle (Fig 3e), but not in other tissues (Fig. S2). Within the hypothalamic-pituitary-gonadal axis (HPG axis), endogenous Lin28a is normally only expressed in thepituitary and ovary. Despite over-expression of Lin28a in the hypothalamus and ovary (Fig.3a), we found no decrease of let-7a or let-7g in the HPG axis, demonstrating that Lin28a andlet-7 are uncoupled in some tissues(Fig. 3f).In a separate study, we showed that doxycycline induction of Lin28a expression expandstransit amplifying cell numbers in a cell autonomous manner in the intestinal crypts, blood,and skin (Viswanathan et al., unpublished data), supporting the hypothesis that increasedprogenitor cell proliferation leads to organ and whole body enlargement. Doxycyclineinduction in Lin28a Tg animals leads to rapid death associated with marked gut pathology,thus precluding the analysis of growth phenotypes associated with higher levels of Lin28a.The un-induced Tg mice exhibited thickened skin, larger bones, and proportionally enlargedvisceral organs. Microscopically, we noted hyperplasia of the skin and bone (Fig. 3g), but nodifferences in visceral organ histology (Fig. S2 and S3). Tg livers are 50-60% larger thanlivers from WT littermates (Fig. 1f), and show increased proliferation as assayed by Ki-67,but no increase in cell size (Fig S3a-c). Skeletal muscle cell diameters were also the same(Fig. S3c). To determine if Lin28a, LIN28B or let-7 could cause cell hypertrophy, weinduced these genes in three doxycycline-inducible ESCs, and observed that cell size wasunaffected (Fig. S3d). These data show that Lin28a mediates organ overgrowth byincreasing cell numbers rather than cell size.The proportional overgrowth suggested that endocrine or metabolic mechanisms might begoverning organismal growth. We failed to observe pituitary adenomas and serum growthhormone and Igf1 were not elevated (Fig. S4), ruling out these etiologies of gigantism.Lin28a has been shown to enhance protein translation of Igf2, whose loss of imprintingcauses a human overgrowth disorder called Beckwith-Wiedemann Syndrome (BWS)[22,23]. We found a ~20-fold increase of Igf2 mRNA in liver and a 2-fold increase inmuscle (Fig. 4a). To determine if Igf2 was driving overgrowth, we crossed Lin28a Tgfemales to Igf2 null males and noted that Lin28a Tg mice lacking Igf2 were still overgrown(Fig. S5a). We confirmed that Igf2 coding mRNA was absent (Fig. S5b), ruling out an Igf2dependent mechanism.Zhu et al.Page 3Nat Genet. Author manuscript; available in PMC 2011 April 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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BWS patients also exhibit hypoglycemia, leading us to test if enhanced glucose utilizationwas contributing to overgrowth [24]. We first found that fasting and fed glucose was lowerin the Tg mice (Fig. 4b-c). Using the glucose tolerance test, we showed that both males andfemales cleared glucose more efficiently (Fig. 4d-e). Using the insulin tolerance test, wefound that Tg mice also had increased insulin sensitivity (Fig. 4f-g). To show that the lowerglucose was due to an increase in peripheral tissue sensitivity rather than an increase insecreted insulin, we showed that insulin levels at fasting and thirty minutes after a glucosechallenge were both lower in Tg mice (Fig. 4h). Consistent with a chronically decreasedneed for insulin, islets were smaller in transgenic animals at two ages (Fig. 4i), furthersupporting the hypothesis that Lin28a mediates enhanced glucose uptake in peripheraltissues. Importantly, the Lin28a Tg mice lacking Igf2 still showed enhanced glucose uptakeand lower fed state glucose (Fig. S5c-d), demonstrating that this phenotype was not Igf2dependent.Because Lin28a is normally expressed in muscle, a major organ for glucose processing, andbecause the Tg mice express 7-fold more Lin28a in muscle, we reasoned that over-expression in muscle might drive increased glucose uptake. We over-expressed Lin28a inC2C12 myoblasts, differentiated them for one week, and found increased 2-deoxy-D-[3H]glucose (2-DG) uptake relative to controls (Fig. 4j). In contrast, shRNA knockdown ofLin28a in C2C12 led to a reduction in labeled glucose uptake (Fig. 4k), demonstrating thatLin28a is both necessary and sufficient for enhanced glucose uptake in these cells. Toidentify mechanisms involved in growth and glucose metabolism, we performed wholegenome mRNA expression analysis in five week old skeletal muscle. Using gene-setenrichment analysis [25,26], we found that expression signatures associated with let-7targets, hypoxia, and the Ras pathway were most significantly upregulated, while six of thefifteen most down-regulated sets involved oxidative phosphorylation or mitochondrial genes(Fig. 4). We hypothesized that increased glucose uptake, increased activity of hypoxiapathways and decreased oxidative phosphorylation represented an increase in glycolyticmetabolism. To test this, we measured serum lactate and found that Lin28a Tg micegenerated more of this glycolytic metabolite than WT controls after glucose challenge (Fig.4m). Together, these data suggest that over-expression of Lin28a in skeletal muscle altersthe metabolic state of the tissue, driving enhanced glucose uptake and favoring glycolyticmetabolism. As the M2 isoform of pyruvate kinase (PKM2) drives glycolysis in embryosand cancer cells [27,28], we asked if Lin28a might be activating Pkm2 in vivo. However, noPkm2 RNA or protein was detected in skeletal muscle or liver of adult WT or Tg animals,excluding Pkm2 expression as a mechanism for increased glycolysis in the Lin28a Tg mice(data not shown).Because GWAS-identified polymorphisms are typically associated with modest changes ingene or cis-regulatory activity, it is difficult to ascertain whether SNPs are associated withgain or loss-of-function effects. In contrast, knockout or transgenic mice often demonstrateeither dramatic phenotypes like lethality or no phenotype at all, making it difficult to modelhuman genetic variation. When induced by doxycycline, the Lin28a Tg mice succumb togene hyperfunction. In the un-induced state, phenotypes associated with variation at thehuman LIN28B locus and in the loci of let-7 target genes were observed due to “leaky”expression that modestly alters Lin28a and let-7 levels. Although Lin28a also has let-7independent functions, the fact that the let-7 targets DOTL1, HMGA2, and CDK6 are alsoassociated with taller stature suggests that LIN28B acts through let-7 to affect height [29].Murine knockouts of many let-7 targets such as Hmga2, Igf2bp1, and Myc are runted,suggesting that larger body size could result from reduced let-7 suppression of its growthpromoting targets [14-18]. In this mouse model, increased Lin28a in muscle results in amodest decrease of let-7 and a global increase in let-7 target gene expression (Fig. 4l). In C.elegans, loss of lin-28 results in precocious vulval differentiation and prematureZhu et al.Page 4Nat Genet. Author manuscript; available in PMC 2011 April 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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developmental progression [30,31], while loss of let-7 leads to reiteration of larval stagesand delayed differentiation [32]. We show that Lin28a gain of function also leads to a delayin murine puberty. Although increased body size and pre-pubescent growth rate arecorrelated with earlier menarche in humans [33], the LIN28B alleles associated with latermenarche were also linked to taller stature [2-5]. Our model predicts that hyperfunction ofLIN28B in humans contributes to increased height and later menarche, and indicate thatsome heterochronic functions of this pathway are conserved from worm to human.In heterochronic fashion, Lin28a may also coordinate the rapid growth and metabolism ofearly embryogenesis and in turn delay the phenotypes associated with adulthood. We foundthat Lin28a causes increased glucose utilization, a mechanism by which it might driveovergrowth in vivo. let-7 target oncogenes such as MYC and KRAS also have profoundeffects on metabolism [34,35], as the anabolic demands of embryos or tumors must be metby a different type of metabolism. In cancer, this use of “aerobic glycolysis” is known as theWarburg Effect [36], a phenomenon that is just starting to be explored in embryonicdevelopment. The Lin28a Tg mouse represents an invaluable tool for sorting out therelationship between metabolism, growth, and developmental timing.MethodsGeneration of transgenic miceFlag-tagged murine Lin28a open reading frame was cloned into pBS plasmid and targetingwas performed into V6.5 ES cells containing M2-rtTA targeted to the Rosa26 locus, aspreviously described [19]. Chimeric mice were generated by injection of ES cells into Balb/c blastocysts, then bred to CD-1 females to generate germline-transmitted pups. The linewas maintained on the CD-1 background and the C57/B6 background by backcrossing 3-6times. For all experiments, littermate controls were used.Quantitative RT-PCRRNA was collected by Trizol, reverse-transcribed using SuperScript II (Invitrogen). mRNAand miRNA expression was measured by quantitative PCR using the Delta-Delta CTmethod as described previously [10].Histological AnalysisTissue samples were fixed in 10% buffered formalin or Bouin’s solution and embedded inparaffin. Immunostaining was performed by using the rabbit anti-Lin28a antibody fromProteintech group (used at 1:300; catalog number 11724-1-AP).ImmunohistochemistrySections of tissues were deparaffinized with xylene and rehydrated with graded series ofethanol (absolute, 95%, 80% and 50%, respectively, and distilled water), followed by twowashes of 5 min each in PBS-T. Antigen retrieval was performed for 20 min in sodiumcitrate buffer (10mM pH 6) at 90-100°C followed by wash with PBST 1× 5 min. Tissuesections were then incubated for 10 min in 3% (v/v) hydrogen peroxide in methanol to blockendogenous peroxidase activity. Sections were then washed for 5 min in PBS-T and blockedat room temperature for 1 h by using 2% normal goat serum, 2% bovine serum albumin(BSA) and 0.1% triton-X in PBS. Tissue sections were then incubated in humidifiedchamber for overnight incubation at 4°C with primary antibody (1/200 in TBST). Sectionswere subsequently washed with PBS-T (3× 5min) and incubated at room temperature for 1 hwith secondary antibody (goat anti rabbit). After a wash with PBS-T (3× 5min), sectionswere incubated with ready to use streptavidin peroxidase (Lab Vision, Fremont, CA) for 10Zhu et al.Page 5Nat Genet. Author manuscript; available in PMC 2011 April 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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min at room temperature and then color was developed by using a DAB kit (Vectorlaboratories, Burlingame, CA). Sections were counterstained with hematoxylin.Glucose and insulin tolerance testsGlucose tolerance tests were accomplished by intraperitoneal injection of glucose (2 gglucose/kg body weight) after an overnight fast (14–18h). Insulin tolerance was tested in 5hour fasted mice by intraperitoneal injection of human regular insulin (0.75 U insulin/kgbody weight; Lilly, Indianapolis, Indiana). Blood glucose was determined with a One TouchBasic glucometer (Lifescan, Milipitas, California). Insulin, GH, and Igf1 levels weremeasured by enzyme-linked immunosorbent assays (Crystal Chem, Chicago, Illinois).Cloning and Plasmid ConstructionMurine Lin28a cDNA was subcloned into pBabe.Puro and pMSCV.Neo retroviral vectors.Lin28a shRNA in lentiviral plasmid was purchased from Sigma-Aldrich(TRCN0000122599). Control shRNA was commercially purchased (SHC002V, Sigma-Aldrich)Viral ProductionFor ecotropic viral production, retroviral plasmid DNA and pCL-Eco were transfected into293T cells in a 1:1 mass ratio and virus harvested after 48h. For VSV-G pseudotypedlentivirus, viral plasmid, lentiviral gag/pol, and VSV-G were transfected in a mass ratio1:0.9:0.1, and virus was harvested after 72 hrs. 1 mL of unconcentrated viral supernatantwas used to infect 50,000 cells. Infected cells were selected on antibiotic prior to subsequentanalysis.Glucose uptake assayC2C12 cells growth, differentiation, and glucose uptake was performed as described in Bertiet al [37].Puberty phenotyping analysisBeginning on the day of weaning, female pups were examined daily, 7 d/wk, between 0800and 1300 h, and the age at VO and concurrent body weight were recorded [38,39]. For uteri/ovary weights, females were sacked at day 26 after birth and organs were weighed. Formating, a male proven to be fertile was placed into a cage with two WT or two Tg females at21 days of age and the first litter date and size were recorded. Estrus analysis was performedas describe previously [40].Statistical analysisData is presented as mean ± SEM, and Student’s t-test (two-tailed distribution, two-sampleunequal variance) was used to calculate P values. Statistical significance is displayed as p <0.05 (one asterisk) or p < 0.01 (two asterisks).MicroarrayRNA from quadriceps muscle from 5 week old WT and Tg mice (n = 2 and 2) wereharvested and processed using RNeasy mini kits from Qiagen. All RNA samples wereDNase treated. The Illumina Ref-8 microarray platform was used to generate data.Gene set analysisGene Set Enrichment Analysis was used to identify gene sets/pathways associated with a setof upregulated or downregulated genes. Gene Set Enrichment Analysis 9 (version 2.0) is anZhu et al.Page 6Nat Genet. Author manuscript; available in PMC 2011 April 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Figure 2. Lin28a Tg mice show a delay in the onset of puberty(a-b) Comparison of the timing of vaginal opening (VO) in WT (blue) and transgenic mice(red) on the CD-1 (a) and C57/B6 backgrounds (b). The cumulative percent of animals withVO is displayed. (c) Weights of WT and Lin28a Tg mice at date of weaning and time ofVO. (d) Uterus/ovary weights measured as a percentage of total body weight at day 26 ofage (n = 10 and 8). (e) The time to first estrus. (f) The time to first litter. (g) The first littersize from these matings. All values represent means +/− SEM (*, p < 0.05; **, p <0.01) andthe numbers of mice (n) are shown in the chart or noted in the legend.Zhu et al.Page 10Nat Genet. Author manuscript; available in PMC 2011 April 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript